The role of TLR4 in photoreceptor {alpha}a crystallin upregulation during early experimental autoimmune uveitis

Abstract

Purpose. Previous studies indicate that the upregulation of alphaA crystallin prevents photoreceptor mitochondrial oxidative stress-mediated apoptosis in experimental autoimmune uveitis (EAU). In this study, the role of TLR4 was investigated in the upregulation of alphaA crystallin in the retinas of animals with EAU. Methods. TLR4(-/-), iNOS(-/-), TNF-alpha(-/-), MyD88(-/-), wild-type (WT) control (C57BL/6), and nude mice (B6.Cg-Foxn1(nu)) were immunized with IRBP mixed with complete Freund's adjuvant; eyes were enucleated on day 7 after immunization. Real-time polymerase chain reaction was first used to detect upregulated inflammatory cytokines and alphaA crystallin in retinas with EAU; confirmed with Western blot analysis, and the site of upregulation was localized by immunohistochemistry. Oxidative stress was localized using 8-OHdG, and TUNEL staining was used to detect apoptosis. Results. In early EAU, increased expression of TNF-alpha, iNOS, and alphaA crystallin genes were detected in the retinas of WT mice, whereas such upregulation was absent in TLR4-deficient mice (P < 0.001). alphaA Crystallin was not elevated in MyD88(-/-), TNF-alpha(-/-), and iNOS(-/-) mice with EAU. Immunostaining revealed TNF-alpha, iNOS, and alphaA crystallin localization in the photoreceptor inner segments and outer plexiform layer in the WT controls with EAU; but such staining was absent in TLR4-deficient mice with EAU. 8-OHdG staining showed oxidative stress in the photoreceptors in WT mice with EAU and there was no apoptosis. Conclusions. TLR4 plays an important role in the upregulation of alphaA crystallin through the interaction of MyD88 and the subsequent generation of TNF-alpha and iNOS in the EAU retina. Such crystallin upregulation may prevent oxidative stress-mediated apoptosis of photoreceptors in uveitis.

Figure 1.

(A) Marked upregulation of TNF-α, iNOS, and αA crystallin were found in WT mice with EAU retinas compared with nude mice with EAU, TLR4^−/− mice with EAU, and nonimmunized controls. Upregulation of TNF-α, iNOS, and αA crystallin was also found in nude mice with EAU compared with TLR4^−/− mice with EAU and nonimmunized controls. TNF-α, iNOS, and αA crystallin were all downregulated in TLR4^−/− mice with EAU compared with nonimmunized WT controls. (B) TNF-α, iNOS, and αA crystallin were not upregulated in MyD88^−/− mice. iNOS and αA crystallin were not elevated in TNF-α^−/− mice. Similarly, there was no upregulation of αA crystallin or TNF-α in iNOS^−/− mice. Gene expression of TNF-α, iNOS, and αA crystallin during early EAU in C57BL/6 (WT), B6.Cg-Foxn1^nu (nude), TLR4^−/−, MyD88^−/−, TNF-α^−/−, and iNOS^−/− mice by real-time PCR analysis using gene-specific primers and normalized to GAPDH. The relative multiple of change in mRNA expression was determined using the 2^-ΔΔCt method. *P < 0.05; **P < 0.001.

Figure 2.

During early EAU, TNF-α, αA crystallin, and iNOS were significantly upregulated compared with both nonimmunized WT control and TLR4^−/− mice with EAU (A, B). There was no upregulation of αA crystallin in iNOS^−/− or in TNF-α^−/− mice in early EAU (C). There was no significant increase in the expression of αA crystallin in the mice treated with CFA and pertussis toxin (D; A, WT control; B, WT injected with pertussis toxin). Equal amounts of total retinal proteins from day 7 WT control, WT EAU, TLR4^−/−, TNF-α^−/−, and iNOS^−/− mice with EAU and WT mice injected with CFA and pertussis toxin were separated on a 15% SDS-polyacrylamide gel (7.5% gel for iNOS). Protein bands were transferred to a nitrocellulose membrane and probed with monoclonal TNF-α, polyclonal anti–αA-crystallin, and polyclonal anti-iNOS as the primary antibodies and with corresponding secondary antibodies tagged with horseradish peroxidase. TNF-α was detected at the molecular mass indicated (∼15 kDa), αA crystallin was detected at ∼20 kDa protein, and iNOS was detected at ∼130 kDa protein. Equal protein loading was confirmed by reprobing blots with monoclonal antibody to β-actin. There was a significant decrease in αA crystallin in the day 7 EAU retina of TLR4^−/− mice compared with the WT control retina. (B) Densitometry measurements show a threefold decrease in TNF-α, a 13-fold decrease in αA crystallin, and a twofold decrease in iNOS protein in TLR4^−/− mice with early EAU compared with WT EAU mice. There was also a ninefold decrease in αA crystallin in TLR4^−/− mice with early EAU compared with WT control. *P < 0.05; **P < 0.001; ***P < 0.0001.

Figure 3.

Immunofluorescence localization of αA crystallin and iNOS in the retina. Tissues were labeled using polyclonal antibody against αA-crystallin and monoclonal anti-iNOS (primary antibodies) and against Cy-2–conjugated donkey anti-rabbit IgG and Texas Red dye–conjugated donkey anti-mouse IgG, respectively (secondary antibodies). (A) Immunostaining of nonimmunized C57BL/6 (WT) mice showed αA crystallin localized in the outer plexiform layer and the photoreceptor inner segments. Nonimmunized TLR4^−/− mice also showed very weak staining in these retinal layers. In both nonimmunized WT and TLR4^−/− mice, there was no detectable amount of iNOS staining. (B) Immunostaining of WT mice EAU day 7 after immunization revealed intense staining of iNOS and αA crystallin in the photoreceptor inner segments and outer plexiform layer; however, minimal staining was present in the inner segments and outer plexiform layer in TLR4^−/− mice with EAU.

Figure 4.

Immunofluorescence localization of NFκB and TNF-α in the retinas of mice with EAU day 7 after immunization. Tissues were labeled using polyclonal anti-NFκB and monoclonal antibody against TNF-α (primary antibodies) along with Cy-2–conjugated donkey anti–rabbit IgG and Texas Red dye–conjugated donkey anti–mouse IgG, respectively (secondary antibodies). Nonimmunized WT and nonimmunized TLR4^−/− mice showed no NFκB or TNF-α staining in the retina. During early EAU, however, WT mice showed NFκB and TNF-α staining in the inner segments of the photoreceptors and the outer plexiform layers, whereas such staining was absent in TLR4^−/− mice.

Figure 5.

Immunofluorescence localization of 8-OHdG in the retina. Tissues were labeled using polyclonal antibody against 8-OHdG (primary antibody) and Texas Red dye–conjugated donkey anti–goat IgG (secondary antibody). No 8-OHdG staining was present in the nonimmunized WT or TLR4^−/− mice. In the WT mice with EAU (day 7 after immunization), intense OHdG staining was detected in the inner segment of the photoreceptor layer, inner nuclear layer, outer plexiform layer, and inner plexiform layer; such staining was markedly reduced in the inner segments and outer plexiform layer and was absent in the remainder of the retina in the TLR4^−/− mice during early EAU.

Figure 6.

Both the negative control and the nonimmunized WT control showed no apoptosis. TUNEL-positive cells were also not detected in WT and TLR4^−/− during early EAU. In contrast, numerous apoptotic cells were seen in positive control retinas treated with DNase I. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; IPL, inner plexiform layer.